PROJECT SUMMARY: The development of cell and animal model-based systems for direct, efficient, titratable and reversible degradation of target proteins Our collective endeavor to determine the functions of the ~ 25,000 genes encoded within the human and mouse genomes has heavily relied on targeted gene knockout or RNA interference (RNAi) knockdown technologies. Despite these approaches being important for scientific progress they are hindered by a number of limitations and caveats, the most significant being that these technologies do not directly target the protein. To alleviate these limitations, we propose to introduce an auxin induced degron (AID) system in human and mouse ESCs using CRISPR/Cas9 technology. The AID technology will enable direct, efficient, titratable and reversible degradation of a target protein. My laboratory studies the Structural Maintenance of Chromosomes (SMC) complexes in the context of stem cell proliferation, differentiation, mammalian gametogenesis and neurodevelopment. SMC complexes include cohesin, condensin and SMC5/6. Malfunction of SMC complexes causes severe developmental disorders including microcephaly, and SMC mutations are associated with cancer development. We will assess the depletion of the SMC5/6 complex for ?proof-of-principle? experiments with regard to the development of the AID-mediated protein degradation system in human and mouse ESC lines. Furthermore, the mouse ESCs will be used to create an AID mouse model to study SMC5/6 complex functions during mammalian neurodevelopment and gametogenesis. Stem cells are key tools for genetic engineering, development of cell-based therapies and basic research. Introduction of AID system into ESCs opens new perspectives in scientific endeavor for researchers from different fields, working with stem cells, cancer cells, as well as with tissue-specific cell types and organoids. These cell lines and organoids will also be of great benefit to tissue engineers and clinicians. Creation of AID mouse models will allow analysis of essentially any developmental stage, any tissue and cell type of interest. This model will result in the development of new approaches to analyze embryogenesis and organogenesis, stem-cell niche interactions and fate decisions. As the AID system is titratable, it can be optimized to better emulate human diseases. Furthermore, this technology will be adaptable to high-throughput genetic and therapeutic screens to study stem cell homeostasis and differentiation capacity, together with any cellular event or feature of interest.